Investigators from multiple departments at the Georgia Institute of Technology / Emory University School of Medicine, from the University of Illinois at Urbana Champaign, and from the Southern Illinois University School of Medicine propose a multidisciplinary project to develop a microfabricated neuronal interfacing system (uNIS) for advanced interfacing to three-dimensional neural tissue in vitro, and to apply this technology to the study of plasticity and injury in neuronal networks. The specific aims of the project are formulated around the development and integration of a set of technological advances that facilitate the study of three-dimensional cell cultures and slices: (1) vertical towers with connecting crossbridges that provide infrastructure for the formation of structured networks as well as for the electrical connectivity; (2) the inclusion of microfluidic channels into these towers to selectively inject nutrients and trophic factors and to apply chemical stimulation to localized regions of the tissue; and (3) the design of integrated circuits that amplify, multiplex, and process the large number (>1000) of signals generated in the uNIS and that facilitate simultaneous recording and stimulation. This technology will be applied directly to the study of three-dimensional, in vitro neural tissue with a particular focus on hypotheses addressing the development of functional networks of neurons, the role of plasticity in these networks, and the study of injury and its effects on network behavior. During the first year of the grant, prototypes of the microtowers, microfluidic structures, and electronics will be developed and integrated with neural tissue. The remainder of the five-year grant will focus on the refinement and testing of the technology and on a set of biological experiments that validate the approach and test the proposed hypotheses. The successful completion of this project will result in the creation of a technological framework for studying a wide variety of cells (neural and non-neural) as well as experimental data that would be inaccessible without this three-dimensional technology.